Islet-1Induced C3H10T1/2Cells Into Myocardial Cells And Relation Of The Histone Acetylation Of Cardic-specific Genes Studies

ObjectivePreliminary study of our group found that build high expression of Islet-1(insulin gene the enhancer binding protein-1, insulin enhanced the binding factor)lentiviral vectors can induce C3H10T1/2stem cells to cardiomyocyte-like cell-specific differentiation, Cardiac-specific protein genes Gata4, of Nkx2.5, Mef2c expression increased。Of Islet-1, the means by which to make these cardiac-specific protein gene expression increased also unclear. This article from the apparent point of view of the genetic histone acetylation, to discuss of Islet-1by raising the level of histone acetylation in myocardial gene to promote its increased expression, and clear the group of Islet-1and cardiac-specific genes the mutual relations of histone acetylation.Methods and materialsRecovery of C3H10T1/2cells containing10%fetal bovine serum in DMEM high glucose medium cultured in a CO2incubator until the cells covered the passage of the1:3ratio in the bottom of about80to90percent. Grown to a density of60%of the C3H10T1/2cells Moi=20by adding the appropriate amount of virus, while adding Polybrene to the total concentration of8mg/L1. Grouping of cells:①C3H10T1/2cell group,②negative control group infected with empty viral vector C3H10cell group,③the experimental group showed high expression of Islet-1lentiviral vector infection of C3H10T1/2cells.2. Experimental methods1) Fluorescence quantitative polymerase chain reaction (quantitative polymerase chain reaction, the Q-PCR) to verify that the lentiviral vector infection of Islet-1gene expression by western blot analysis detected three groups of acetylated histone H3expression. Using rt-Pcr to detect myocardial gene Gata4, Nkx2.5, Mef2c expression of high expression of Islet-1peak time, and at this point using the chromatin immunoprecipitation (the Chromatin immunoprecitation, ChIP) to detcet the experimental group and the expression of other groups of CHIP-level acetylation histone H3antibody immunoprecipitation with its combination of DNA。2) Observing the effection of40umol/L,80umol/L,120umol/L,160umol/L concentration of EGCG (deacetylase inhibitor) to C3H10T1/2cells growth。Western blot validation of these three concentrations of EGCG on the acetylated histone H3expression, Rt-Pcr verify EGCG inhibition of the strongest time on the cardiac-specific genes, Using chromatin co-immunoprecipitation techniques and the CHIP level of Islet-1antibody immunoprecipitation combination of DNA then compared the expression of each groups。RESULTS1. Q-PCR results showed that infection of Islet-llentiviral vector, the Islet-1gene expression increased, Western blot results showed that the experimental group acetylated histone H3expression increased (P <0.05), in two weeks of infection of lentiviral vector, Myocardial gene expression peaked. Chromatin immunoprecipitation results show the experimental group combined acetylated H3antibody cardiac-specific protein genes Gata4, of Nkx2.5, Mef2c of the DNA content higher than the other two groups (P<0.05).2. We found that160umol/L concentration of EGCG make the cells all dead, so select40umol/L,80umol/L,120umol/L concentration of EGCG to test。Western blot validation120umol/L concentration of EGCG have the strongest inhibition role in the expression of acetylated histone H3(P<0.05). Q-PCR results showed that it’s strongest inhibition on myocardial gene after add EGCG3hours(P <0.05). Infected with lentiviral vectors1w, the Islet-1gene expression in a large number of increased and after two weeks it expression stable, chromatin immune precipitation technique showed that compared with the experimental group which did not add the EGCG, the EGCG experimental group showed that Islet-1combined with the cardiac-specific protein genes Gata4, of Nkx2.5, Mef2c of the DNA expression decreased (P<0.05).Conclusions1. Islet-1induced C3H10T1/2cells into myocardial cells and have a promoting role in the histone acetylation of cardic-specific genes.2. Reduce the level of cardiac-specific protein gene histone acetylation may affect the combination of Islet-1DNA sites...